This proposal presents a combination of high-throughput experimental and computational approaches centered on the identification of functional transcriptional elements in the human genome. The proposed research is consistent with ENCODE's requirement for comprehensive analyses of all target regions, and will be scalable to the whole genome level. In addition, the results can easily be transferred to the ENCODE database and other public databases for sharing with the other participants of the consortium. In Aim 1, promoters of all the genes in the ENCODE targets will be comprehensively identified and tested for activity by a screening scheme and a selection scheme. In Aim 2, a powerful selection method will be used to identify enhancers in the target regions, and their properties will be characterized. In Aim 3, a combination of chromatin immunoprecipitation and microarray hybridization will be used to identify cis-acting binding sites that are occupied by 12 general transcription factors and chromatin proteins in 24 human cell lines. Sequences of bound segments, along with sequences from promoters and enhancers identified in Aims 1 and 2, will be examined intensively with statistical computational methods to identify motifs that are the likely recognition sites for the proteins. In Aim 4, all the pan-mammalian DNA elements that are evolutionarily constrained in the target regions will be identified by using advanced computational, evolutionary, and statistical methodology, producing not only a complete parts list, but also high-resolution, quantitative estimates of evolutionary constraint within each element. In Aim 5, human variation will be identified m a comprehensive sample of the most constrained functional elements by determining the sequence of the element in 48 humans from diverse backgrounds. This sequence data will help answer the question of whether SNPs in noncoding functional elements are as important for human genetics as are cSNPs, and whether the importance of an element for human genetics can be predicted on the basis of evolutionary comparisons. This proposed work constitutes a comprehensive characterization of an important subset of all functional elements in the target regions.